Protein-dependent Membrane Interaction of A Partially Disordered Protein Complex with Oleic Acid: Implications for Cancer Lipidomics

Abstract

Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA.

Direct observation of alpha-lactalbumin, adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures

“…In the context of protein-related diseases, unraveling them is fundamental in order to better understand both the amyloid formation in vivo and the related toxicity [30]. To this aim a great attention has been recently addressed to the interaction between intrinsically disordered regions of proteins and membrane, which may be involved both in protein function and dysfunction in cellular environment [31][32][33].…”

“…This is a compact denatured state partially retaining the native secondary structure but lacking of a well-defined tertiary structure [39,42] that is known to rapidly insert into the lipid membrane [29,43,44]. Moreover, this state is characterized by a high conformational flexibility [42,45] and thus represents a good model for monitoring protein-membrane interactions in conditions where a stable global protein fold is missing [11,32,46].…”

“…A great number of experiments were performed on α-La interaction with lipid bilayers, using both small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) as model membranes [13,24,26,41,[43][44][45][47][48][49]. Besides being an excellent model system for studying protein-membrane interactions and describing specific mechanisms for amphitrophic protein behavior [11], the specific interest in the analysis of α-La-membrane interaction is mainly based on the fact that the bovine and human variant of this protein can alter its biological function and gain a tumoricidal property if partially unfolded and bound to oleic acid [32,[50][51][52]. These complexes are named HAMLET (human α-La made lethal to tumor cells) and BAMLET (bovine α-La made lethal to tumor cells) [50,51] and their interaction with membrane has been shown to be crucial to trigger tumor cell death [32,53,54].…”

“…Besides being an excellent model system for studying protein-membrane interactions and describing specific mechanisms for amphitrophic protein behavior [11], the specific interest in the analysis of α-La-membrane interaction is mainly based on the fact that the bovine and human variant of this protein can alter its biological function and gain a tumoricidal property if partially unfolded and bound to oleic acid [32,[50][51][52]. These complexes are named HAMLET (human α-La made lethal to tumor cells) and BAMLET (bovine α-La made lethal to tumor cells) [50,51] and their interaction with membrane has been shown to be crucial to trigger tumor cell death [32,53,54]. Nevertheless, results remain controversial and a clear unifying picture of membrane and protein structural changes related to the formation of membrane-protein complexes is not clear.…”

Role of Charge and Hydrophobicity in Liprotide Formation: A Molecular Dynamics Study with Experimental Constraints

“…These buried hydrophobic residues are often involved in the interactions with OA in the final liprotide structures and further demonstrate why unfolding of the protein is important. Earlier MD simulations by Chaudhuri and co‐workers suggested that residues from all over aLA interact with OA when aLA is folded . However, Chaudhuri and co‐workers only used a small number of OA molecules, leading to full exposure of individual OA molecules and facilitating interactions between the hydrophobic tail of OA and aLA.…”